Method for increasing the activity of acid-β-galactosidase

ABSTRACT

Method for enhancing in a mammalian cell the activity of an enzyme associated with Gaucher Disease by administering a competitive inhibitor of glucocerebrosidase in an amount effective to enhance the activity of the enzyme. Preferred compounds for use in the method are imino sugars and related compounds. In particular, C8-12-alkyl derivatives of N-alkyl-deoxynojirimycin, isofagomine compounds, and calystegine compoiunds are effective to enhance glucocerebrosidase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/989,258,filed on Nov. 16, 2004, now U.S. Pat. No. 7,141,582, which is acontinuation of application Ser. No. 10/304,395, filed on Nov. 26, 2002,now U.S. Pat. No. 6,916,829, which is a continuation of application Ser.No. 09/948,348, filed on Sep. 7, 2001, now U.S. Pat. No. 6,599,919,which is a continuation of application Ser. No. 09/604,053, filed onJun. 26, 2000, now U.S. Pat. No. 6,583,158, which is acontinuation-in-part of application Ser. No. 09/087,804, filed on Jun.1, 1998, now U.S. Pat. No. 6,274,597. Each of these prior references arehereby incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of enhancing mutant enzyme activitiesin lysosomal storage disorders by administration of competitiveinhibitors of the enzymes, in particular imino sugars and relatedcompounds.

2. Background Information

Proteins are synthesized in the cytoplasm, and the newly synthesizedproteins are secreted into the lumen of the endoplasmic reticulum (ER)in a largely unfolded state. In general, protein folding is an eventgoverned by a principle of self assembly. The tendency of proteins tofold into their native (active) conformation is contained in their aminoacid sequences (1). In vitro, the primary structure folds into secondarystructures of α-helices and β-sheets coupled with hydrophobic collapsein the formation of a biologically active tertiary structure which alsogains increased conformational stability. However, the folding ofprotein in vivo is rather complicated, because the combination ofambient temperature and high protein concentration stimulates theprocess of aggregation, in which amino acids normally buried in thehydrophobic core interact with their neighbours non-specifically. Toavoid this problem, protein folding is usually facilitated by a specialgroup of proteins called molecular chaperones which prevent nascentpolypeptide chains from aggregating, and bind to protein so that theprotein refolds in the active state (2).

Molecular chaperones are present in virtually all types of cells and inmost cellular compartments. Some are involved in the transport ofproteins and in supporting cells to survive under stresses such as heatshock and glucose starvation. Among the molecular chaperones (3-6), BIP(immunoglobulin heavy-chain binding protein, Grp78) is the bestcharacterized chaperone of the ER (7). Like other molecular chaperones,BIP interacts with many secretory and membrane proteins within the ERthroughout their maturation, although the interaction is normally weakand short-lived when the folding proceeds smoothly. Once the nativeprotein conformation is achieved, the molecular chaperone no longerbinds. However, the interaction between BIP and those proteins that failto fold, assemble or be properly glycosylated becomes stable, andusually leads to degradation of these proteins through the ubiquitinpathway. This process serves as a “quality control” system in the ERwhich ensures that only properly folded and assembled proteins aretransported to the Golgi complex for further maturation, and thoseimproperly folded proteins are retained for subsequent degradation (8).

In many hereditary disorders, mutant gene products are structurallyaltered and may not fold correctly, signalling the quality controlsystem to retain and degrade them in situ. This process may contributesignificantly to the protein deficiency, although the function of theprotein may have been only partially impaired (9-12). For example, themost common mutation in cystic fibrosis, a deletion of phenylalanine-508(ΔF508) in the CFTR protein which functions as a chloride channel in theplasma membrane, results in misfolding and retardation of the ΔF508-CFTRprotein in the ER, and subsequent degradation by the cytosolicproteasome system (13-14), even though it retains almost full biologicactivity when inserted into plasma membranes (15). The list of diseasescaused by mutations that alter protein folding is increasing, and itincludes α₁-antitrypsin deficiency (16-17), familialhypercholesterolemia (18), Alzheimer's disease (18a), Marfan syndrome(19), osteogenesis imperfecta (20), carbohydrate-deficient glycoproteinsyndrome (21), and Maroteaux-Lamy syndrome (22).

Lysosomal storage disorders are a group of diseases resulting from theabnormal metabolism of various substrates, including glycosphingolipids,glycogen, mucopolysaccharides and glycoproteins. The metabolism of exo-and endogenous high molecular weight compounds normally occurs in thelysosomes, and the process is normally regulated in a stepwise processby degradation enzymes. Therefore, a deficient activity in one enzymemay impair the process, resulting in an accumulation of particularsubstrates. Most of these diseases can be clinically classified intosubtypes: i) infantile-onset; ii) juvenile-onset; or iii) late-onset.The infantile-onset forms are often the most severe usually with noresidual enzyme activity. The later-onset forms are often milder withlow, but often detectable residual enzyme activity. The severity of thejuvenile-onset forms are in between the infantile-onset and late-onsetforms. Table 1 contains a list of a number of known lysosomal storagedisorders and their associated defective enzymes. In the adult-onsetforms of lysosomal storage disorders listed in Table 1, certainmutations cause instability of the encoded protein.

TABLE 1 Lysosomal storage disorders. Lysosomal storage disorderDefective enzyme Pompe disease Acid α-glucosidase Gaucher disease Acidβ-glucosidse, or glucocerebrosidase Fabry disease α-Galactosidase AG_(M1)-gangliosidosis Acid β-galactosidase Tay-Sachs diseaseβ-Hexosaminidase A Sandhoff disease β-Hexosaminidase B Niemann-Pickdisease Acid sphingomyelinase Krabbe disease Galactocerebrosidase Farberdisease Acid ceramidase Metachromatic leukodystrophy Arylsulfatase AHurler-Scheie disease α-L-Iduronidase Hunter diseaseIduronate-2-sulfatase Sanfilippo disease A Heparan N-sulfataseSanfilippo disease B α-N-Acetylglucosaminidase Sanfilippo disease CAcetyl-CoA: α-glucosaminide N- acetyltransferase Sanfilippo disease DN-Acetylglucosamine-6-sulfate sulfatase Morquio disease AN-Acetylgalactosamine-6-sulfate sulfatase Morquio disease B Acidβ-galactosidase Maroteaux-Lamy disease Arylsulfatase B Sly diseaseβ-Glucuronidase α-Mannosidosis Acid α-mannosidase β-Mannosidosis Acidβ-mannosidase Fucosidosis Acid α-L-fucosidase Sialidosis SialidaseSchindler-Kanzaki disease α-N-acetylgalactosaminidase

In their earlier filed patent application (U.S. application Ser. No.09/087,804), the present inventors proposed a novel therapeutic strategyfor Fabry disease, a lysosomal storage disorder caused by deficientlysosomal α-galactosidase A (α-Gal A) activity in which certainmutations encoded mutant proteins which have folding defects. Theapplication presented evidence demonstrating that1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of α-GalA, effectively increased in vitro stability of a mutant α-Gal A (R301Q)at neutral pH and enhanced the mutant enzyme activity in lymphoblastsestablished from Fabry patients with the R301Q or Q279E mutations.Furthermore, oral administration of DGJ to trangenic mice overexpressinga mutant (R301Q) α-Gal A substantially elevated the enzyme activity inmajor organs (24).

The principle of this strategy is as follows. Since the mutant enzymeprotein appears to fold improperly in the ER where pH is neutral, asevidenced by its instability at pH 7 in vitro (25), the enzyme proteinwould be retarded in the normal transport pathway (ER→Golgiapparatus→endosome→lysosome) and subjected to rapid degradation. Incontrast, an enzyme protein with a proper folding conformation could beefficiently transported to the lysosomes and remain active, because theenzyme is more stable below pH 5. Therefore, a functional compound whichis able to induce a stable molecular conformation of the enzyme isexpected to serve as a “chemical chaperone” for the mutant protein tostabilize the mutant protein in a proper conformation for transport tothe lysosomes. Some inhibitors of an enzyme are known to occupy thecatalytic center of enzyme, resulting in stabilization of itsconformation in vitro, they may also serve as “chemical chaperones” toenforce the proper folding of enzyme in vivo, thus rescue the mutantenzyme from the ER quality control system. It is noted that while thisis believed to be the mechanism of operation of the present invention,the success of the invention is not dependent upon this being thecorrect mechanism.

SUMMARY OF THE INVENTION

The present inventors have unexpectedly found that potent competitiveinhibitors for enzymes associated with lysosomal storage disordersenhance the activity of such enzymes in cells when administered atconcentrations lower than that normally required to inhibit theintracellular enzyme activity. The effect is particularly significant oncertain defective or mutant enzymes, but also occurs in cells containingthe normal enzyme type.

Accordingly, it is one object of the present invention to provide amethod of preventing degradation of mutant enzymes associated withlysosomal storage diseases in mammalian cells, particularly in humancells.

It is a further object of the invention to provide a method of enhancingthe activity of enzymes associated with lysosomal storage disease inmammalian cells, particularly in human cells. The method of the presentinvention enhance the activity of both normal and mutant α-Gal A,particularly of mutant α-Gal A which is present in certain forms ofFabry disease. The methods of the present invention also enhance theactivity of certain mutant β-galactosidase and glucocerebrosidase andare expected to be useful in other lysosomal storage diseases, includingthose listed in Table 1.

In addition, the methods of the invention are also expected to be usefulin nonmammalian cells, such as, for example, cultured insect cells andCHO cells which are used for production of α-Gal A for enzymereplacement therapy.

It is yet a further object of the invention to provide a method oftreatment for patients with lysosomal storage disorders such as thoselisted in Table 1.

Compounds expected to be particularly effective for Fabry disease in themethods of the invention are galactose and glucose derivatives having anitrogen replacing the oxygen in the ring preferably galactosederivatives such as 1-deoxygalactonojirimycin and4-epi-α-homonojirimycin. The term “galactose derivative” is intended tomean that the hydroxyl group at the C-3 position is equatorial and thehydroxyl group at the C-4 position is axial, as represented, forexample, by the following structures:

wherein R₀ represents H, methyl or ethyl; R₁ and R₁′ independentlyrepresent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g.,CH₂OH); R₂ and R₂′ independently represent H, OH or alkyl group (n=1-8).

Other specific competitive inhibitors for α-galactosidase, such as forexample. calystegine A₃ and B₂, and N-methyl derivatives of thesecompounds should be useful in the method of the invention. Thecalystegine compounds can be represented by the formula

wherein for calystegine A₃: R₀═H, R₂═R₂′═H, R₄═OH. R₄′═R₇═H; forcalystegine B₂: R₀═H. R₂═OH, R₂′═R₄′═H, R₄═OH, R₇═H; forN-methyl-calystegine A₃: R₀═CH₃, R₂═R′═H. R₄═OH, R₄′═R₇═H; forN-methyl-calystegine B₂: R₀═CH₃, R₂═OH, R₂′═R₄′═H. R₄═OH. R₇=H.

Administration of a pharmaceutically effective amount of a compound offormula

wherein R₀ represents H, methyl or ethyl; R₁ and R₁ independentlyrepresent H, OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g.,CH₂OH); R₂ and R₂′ independently represent H, OH or alkyl group (n=1-8);R₄ and R₄′ independently represent H, OH, or a compound selected fromthe group consisting of α-allo-homonojirimycin,α-galacto-homonojirimycin, β-1-C-butyl-deoxygalactonojirimycin,calystegine A₃, calystegine B₂ and their N-alkyl derivatives willalleviate the symptoms of Fabry disease by increasing the residualenzyme activity in patients suffering from Fabry disease.

Compounds expected to be particularly effective forG_(M1)-gangliosidosis in the methods of the invention are galactosederivatives having a nitrogen replacing the oxygen in the ring or anitrogen at the same position of the anomeric position of a pyranosering, preferably galactose derivatives such as 4-epi-isofagomine and1-deoxygalactonojirimycin.

Administration of a pharmaceutically effective amount of a compound offormula

wherein R₀ represents H, methyl or ethyl, R₁ and R₁′ independentlyrepresent H. OH, a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g.,CH₂OH): R₂ and R₂′ independently represent H. OH or alkyl group (n=1-8);or a compound selected from the group consisting of 4-epi-isofagomine.and 1-deoxygalactonojirimycin and their N-alkyl derivatives willalleviate the symptoms of G_(M1)-gangliosidosis by increasing theresidual β-galactosidase activity in patients suffering fromG_(M1)-gangliosidosis.

Compounds expected to be particularly effective for Gaucher disease inthe methods of the invention are glucose derivatives having a nitrogenreplacing the oxygen in the ring or a nitrogen at the same position ofthe anomeric position of a pyranose ring, preferably glucose derivativessuch as N-dodecyl-deoxynojirimycin and isofagomine. The term “glucosederivative” is intended to mean that the hydroxyl groups at the C-3 andC-4 positions are equatorial as represented, for example, by thefollowing structures:

wherein R₀ represents H, alkyl chain (n=8-12); R₀′ represents H, astraight chain or branched saturated or unsaturated carbon chaincontaining 1-12 carbon atoms, optionally substituted with a phenyl,hydroxyl or cyclohexyl group; R₁ and R₁′ independently represent H, OH,a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g., CH₂OH); R₂ andR₂′ independently represent H, OH or alkyl group (n=1-8).

Other specific competitive inhibitors for β-glucosidase such as forexample, calystegine A₃, B₁, B₂ and C₁ and their derivatives of thesecompounds should be useful in the method of the invention. Thecalystegine compounds can be represented by the formula

wherein for calystegine A₃: R₀═H, R₂═R₂′═H, RA=OH, R₄′═R₇═H; forcalystegine B₁: R₀═H, R₂═R₂′═R₄′═H, R₄═OH, R₇═OH; for calystegine B₂:R₀═H, R₂═OH, R₂′═R₄′═H, R₁═OH, R₇═H; for calystegine C₁: R₀═H, R₂═OH,R₂′═H, R₄═OH, R₄′═H, R₇═OH.

Administration of a pharmaceutically effective amount of a compound offormula

wherein R₀ represents H, alkyl chain (n=8-12); R₀′ represents H, astraight chain or branched saturated or unsaturated carbon chaincontaining 1-12 carbon atoms, optionally substituted with a phenyl,hydroxyl or cyclohexyl group; R₁ and R₁′ independently represent H, OH,a 1-4 carbon alkyl, alkoxy or hydroxyalkyl group (e.g., CH₂OH); R₂ andR₂′ independently represent H, OH or alkyl group (n=1-8): or a compoundselected from the group consisting of isofagomine, N-butyl-isofagomine,N-(3-cyclohexylpropyl)-isofagomine, N-(3-phenylpropyl)-isofagomine andN-|(2E,6Z,10Z)-3,7.11-trimethyldodecatrienyl|-isofagomine.N-dodecyl-deoxynojirimycin will alleviate the symptoms of Gaucherdisease by increasing the residual glucocerebrosidase activity inpatients suffering from Gaucher disease. Other competitive inhibitors ofglucocerebrosidase, such as calystegine compounds and N-alkylderivatives thereof should also be useful for treating Gaucher disease.Similarly, known competitive inhibitors of other enzymes associated withlysosomal storage disorders listed in Table 1 will be useful in treatingthose disorders.

Persons of skill in the art will understand that an effective amount ofthe compounds used in the methods of the invention can be determined byroutine experimentation, but is expected to be an amount resulting inserum levels between 0.01 and 100 μM, preferably between 0.01 and 10 μM,most preferably between 0.05 and 1 μM. The effective dose of thecompounds is expected to be between 0.5 and 1000 mg/kg body weight/day,preferably between 0.5 and 100 mg/kg body weight/day, most preferablybetween 1 and 50 mg/kg body weight/day. The compounds can beadministered alone or optionally along with pharmaceutically acceptablecarriers and excipients, in preformulated dosages. The administration ofan effective amount of the compound will result in an increase in thelysosomal enzymatic activity of the cells of a patient sufficient toimprove the symptoms of the disease.

In many lysosomal storage diseases, much of the clinical variability andage of onset can be attributed to small differences in the residualactivity of the affected enzyme (25a). Pseudodeficiency of lysosomalstorage disorders identified as clinically healthy probands withseverely reduced activity (10-20% of normal) of a lysosomal enzymesuggests that a small increase of residual enzyme activity could have alarge effect on the disease (25b). Particularly in Fabry disease, asmall augmentation in enzyme stability resulting in an. increase ofresidual α-Gal A activity is expected to have a significant impact onthe disease, based on the observations on the cardiac variants with 10%residual activity (2). Therefore, a small percentage increase of theresidual enzyme activity may alleviate symptoms of the disease orsignificant delay the development of the disease.

Compounds disclosed herein and other competitive inhibitors of enzymesassociated with lysosomal storage diseases which will be known to thoseof skill in the art will be useful according to the invention in methodsof enhancing the intracellular activity of normal and mutant enzymesassociated with such disorders and treating the disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of inhibitors used in this study.

The numbering of carbons for compounds 2-6, 11-16, 30-32 is the same asin compound 1; the numbering of carbons for compounds 8-10 is the sameas in compound 7; the numbering of carbons for compounds 18-20, 22-26 isthe same as in compound 17; the numbering of carbons for compounds 33-37is the same as in compound 21.

FIG. 2. Stereostructure of α-allo-HNJ (9).

A and B are identical, but viewed from different angles. C is a proposedstereo-structure for α-allo-HNJ (9) deduced from NMR analysis. D is apresumed reaction intermediate for α-galactosidase.

FIG. 3. Lineweaver-Burk plots of DGJ (4) (A), α-allo-HNJ (9) (B),α-galacto-HNJ (10) (C), and β-1-C-butyl-DGJ (16) (D) inhibition of α-GalA.

The increasing concentrations of substrate were used to determine theK_(m) and K_(i) values and the data were plotted as 1/v vs. 1/S. Thecalculated K_(m) for p-nitrophenyl-α-D-galactopyranoside was 0.17 mM.(A) Concentrations of 4 were 0 (●), 0.1 μM (▴), and 0.25 μM (▪). Thecalculated K_(i) value was 0.04 μM. (B) Concentrations of 9 were 0 (●),2.5 μM (▴), and 5 μM (▪). The calculated K_(i) value was 2.6 μM. (C)Concentrations of 10 were 0 (●), 0.1 μM (▴), and 0.25 μM (▪). Thecalculated K_(i) value was 0.17 μM. (D) Concentrations of 16 were 0 (●),10 μM (▴), and 25 μM (▪). The calculated K_(i) value was 16 μM.

FIG. 4. In vitro inhibition (A) and intracellular enhancement (B) ofα-Gal A by inhibitors.

(A) Concentrations for 50% inhibition of α-Gal A (IC₅₀) were determinedwith p-nitrophenyl-α-D-galactopyranoside as the substrate. (B) Eachinhibitor was added to the culture medium of R301Q lymphoblasts at aconcentration of 100 μM. Cells were subsequently incubated for 4 days.After being washed twice with phosphate-buffered saline, theintracellular enzyme activity was determined with 4-MU-α-Gal as thesubstrate.

FIG. 5. Effect of the increasing concentrations of selected inhibitorson α-Gal A activity.

R301Q lymphoblasts were cultured with DGJ (4) (●), α-allo-HNJ (9) (▪),α-galacto-HNJ (10) (▴), or β-1-C-butyl-DGJ (16) (♦) at 1-1000 μM for 4days before being collected for enzyme assay.

FIG. 6. Intracellular enhancement of α-Gal A activity in Fabrylymphoblasts by calystegine compounds.

R301Q lymphoblasts were cultured with calystegine A₃ (17) (A),calystegine B₂ (18) (B), N-methyl calystegine A₃ (19) (C), or N-methylcalystegine B₂ (20) (D) at 100-1000 μM for 4 days before being collectedfor enzyme assay.

FIG. 7. Intracellular enhancement of β-Gal activity inG_(M1)-gangliosidosis fibroblasts.

A, G_(M1)-gangliosidosis fibroblasts were cultured with DGJ (4) at 500μM for 5 days before being collected for enzyme assay. B,G_(M1)-gangliosidosis fibroblasts were cultured with 4-epi-isofagomine(21) at 50 μM for 5 days before being collected for enzyme assay.Strains BGF-1 and BGF-6 were diagnosed as infantile type, and all otherswere juvenile or adult types.

FIG. 8. Intracellular enhancement of β-Glu activity in Gaucherfibroblasts by isofagomine (33).

A, Gaucher fibroblasts (N370S/N370S) were cultured with isofagomine (33)at 1-100 μM for 5 days before being collected for enzyme assay. B, Timecourse of intracellular enhancement of β-Glu activity in Gaucher cellsby isofagomine.

FIG. 9. Intracellular enhancement of β-Glu activity in Gaucherfibroblasts by isofagomine derivatives. Gaucher fibroblasts(N370S/N370S) were cultured with isofagomine derivatives (34-37) at 10or 100 μM for 5 days before being collected for enzyme assay.

FIG. 10. Intracellular enhancement of β-Glu activity in Gaucherfibroblasts by dodecyl-DNJ (31).

A, Gaucher fibroblasts (N370S/N370S) were cultured with dodecyl-DNJ (31)at 0.05-5 μM for 5 days before being collected for enzyme assay. B, Timecourse of intracellular enhancement of β-Glu activity in Gaucher cellscultured in the presence (◯) or absence (●) of dodecyl-DNJ.

FIG. 11. Intracellular enhancement of β-Glu activity in Gaucherfibroblasts by calystegine compounds. Gaucher fibroblasts (L444P/L444P)were cultured with calystegine compounds (17, 18, 23 and 26) at 1-100 μMfor 5 days before being collected for enzyme assay. A, calystegine B₂(18); B, calystegine B₁ (23); C, calystegine A₃ (17); and D, calystegineC₁ (26).

FIG. 12. Structure of iminosugars

DETAILED DESCRIPTION OF THE INVENTION

In the work leading to the present application, the inventors tested aseries of naturally occurring and chemically synthesized novelinhibitors for both in vitro inhibition of normal α-Gal A andintracellular enhancement of a mutant α-Gal A activity with Fabrylymphoblasts to demonstrate that potent competitive reversibleinhibitors of α-Gal A are effective “chemical chaperones” which canstabilize the mutant enzyme and rescue it from degradation. Applicantsnow have tested the chemical chaperone strategy with Gaucher disease andG_(M1)-gangliosidosis, both of which belong to the lysosomal storagedisorder family (26-27), to demonstrate that this therapeutic strategyof using potent competitive inhibitors as chemical chaperones to enhancethe residual enzyme activity in the patient's cells is not limited toFabry disease, and can be applied to Gaucher disease andG_(M1)-gangliosidosis as examples of the principle which can be extendedto to other lysosomal storage disorders as listed in Table 1.

Materials and Methods

Inhibitors The structures of the Inhibitors used in this Invention areShown in FIG. 1.

1-deoxynojirimycin (DNJ) (1) was isolated from the roots of Morus alba(Moraceae) as described previously (28). 1-Deoxymannojirimycin(manno-DNJ) (2) and 1-deoxy-3,4-diepi-nojirimycin (gulo-DNJ) (5) havebeen recently isolated from the barks of Angylocalyx pynaertii(Leguminosae). 1-Deoxy-3-epi-nojirimycin (allo-DNJ) (3) was prepared bythe microbial redox reaction at C-3 of the N-benzyloxycarbonylderivative of DNJ as described previously (29).1-Deoxygalactonojirimycin (DGJ) (4) and 1,2-dideoxy-galactonojirimycin(6) were prepared by the chemical epimerization of the 4-OH group of 1and fagomine, respectively, according to the literature procedure (30).α-Homonojirimycin (α-HNJ) (7), α-homomannojirimycin (α-manno-HNJ) (8),and α-homoallonojirimycin (α-allo-HNJ) (9) were isolated from the wholeplant of Aglaonema treubii (Araceae) as reported previously (31).α-Homogalactonojirimycin (α-galacto-HNJ) (10) was prepared from2,3,4,6-tetra-O-benzyl-D-galactose by way of a Wittig chain extensionand a mercuricyclization according to the literature procedure (33).N-Methyl-DGJ (11) was prepared by treatment of 4 with 37% HCHO and 80%formic acid according to the reference (34), and the N-ethyl (12),N-propyl (13), N-butyl (14) and N-hydroxyethyl (15) derivatives of 4were prepared by treatment with the appropriate alkyl bromide andtriethylamine in DMF. The reaction mixture of N-alkylation wasevaporated in vacuo, and the residual syrup was resolved in MeOH andapplied to an Amberlyst 15 column (H⁺ form), washed with MeOH, elutedwith 0.5 M NH₄OH, and concentrated. N-Alkylated derivatives were finallypurified by Dowex 1X2 (OH⁻ form) and Amberlite CG-50 (NH₄ ⁺ form)chromatography with water as eluent. β-1-C-Butyl-DGJ (16) was isolatedfrom Adenophorae Radix as described previously (35). 4-epi-isofagomine(21), isofagomine (33) and its derivatives (34-37) were chemicallysynthesized as described previously (40).2,5-Dideoxy-2,5-imino-D-mannitol (DMDP, 27),1,4-dideoxy-1,4-imino-D-arabinitol (DAB, 28) were purified from Derrismalaccensis and Morus alba, respectively (29). N-butyl-DNJ (30),N-dodecyl-DNJ (31), castanospermine (29) were from commercial sources.Calystegine A₃ (17), calystegine A₅ (22), calystegine B₁ (23),calystegine B₂ (18), calystegine B₃ (24), calystegine B₄ (25), andcalystegine C₁ (26) were prepared as published method (36, 40a),N-methyl calystegine A₃ (19) and N-methyl calystegine B₂ (20) werechemically synthesized.

Structural characterization of some inhibitors The structuralcharacterization of the inhibitors is determined by mass spectrometryand ¹³C-NMR, and some of the results are presented below.

1-Deoxynojirimycin (DNJ) (1) HRFABMS m/z 164.0923 [M+H]⁺ (C₆H₁₄NO₄requires 164.0923). ¹³C-NMR (100 MHz, D₂O) δ(ppm downfield from internalsodium 3 -(trimethylsilyl)propionate) 51.5 (C-1), 63.3 (C-5), 64.2(C-6), 73.7 (C-2), 74.3 (C-4), 81.2 (C-3).

1-Deoxymannojirimycin (manno-DNJ) (2) HRFABMS m/z 164.0923 [M+H]⁺(C₆H₁₄NO₄ requires 164.0923). ¹³C-NMR(100 MHz, D₂O) δ51.5 (C-1), 63.4(C-5), 63.7 (C-6), 71.3 (C-4), 72.1 (C-2), 77.5 (C-3).

1-Deoxy-3-epi-nojirimycin (allo-DNJ) (3) HRFABMS m/z 164.0922 [M+H]⁺(C₆H₁₄NO₄ requires 164.0923). ¹³C-NMR (100 MHz, D₂O) δ46.9 (C-1), 59.2(C-5), 60.7 (C-6), 72.7(C-2), 73.3 (C-4), 75.0 (C-3).

1-Deoxygalactonojirimycin (DGJ) (4) HRFABMS m/z 164.0921 [M+H]⁺(C₆H₁₄NO₄ requires 164.0923). ¹³C-NMR(100 MHz, D₂O) δ51.9 (C-1), 61.7(C-5), 64.2 (C-6), 70.9 (C-2), 72.1 (C-4), 77.9 (C-3).

1-Deoxy-3,4-diepi-nojirimycin (gulo-DNJ) (5) HRFABMS m/z 164.0921 [M+H]⁺ (C₆H₁₄NO₄ requires 164.0923). ¹³C-NMR (100 MHz, D₂O) δ46.9 (C-1),56.9 (C-5), 63.8 (C-6), 68.3 (C-2), 72.0 (C4),73.0 (C-3).

1,2-Dideoxygalactonojirimycin (6) HRFABMS m/z 148.0972 [M+H]⁺ (C₆H₁₄NO3requires 148.0974). ¹³C-NMR(100 MHz, D₂O) δ30.2 (C-2), 45.6 (C-1), 61.8(C-5), 64.5 (C-6), 70.5 (C-4), 72.7 (C-3).

α-Homonojirimycin (α-HNJ) (7) HRFABMS m/z 194.1025 [M+H]⁺ (C₇H₁₆NO₅requires 194.1028). ¹³C-NMR (100 MHz, D₂O) ,δ56.9 (C-5), 59.1 (C-1′),59.7 (C-1), 64.8 (C-6), 74.4 (C-2), 74.9 (C-4), 77.1 (C-3).

α-Homomannojirimycin (α-manno-HNJ) (8) HRFABMS m/z 194.1026 [M+]⁺(C₇H₁₆NO₅ requires 194.1028). ¹³C-NMR (100 MHz, D₂O) δ58.6 (C-5), 61.4(C-1), 62.2 (C-1′), 63.9 (C-6), 71.4 (C-4), 71.6 (C-2), 74.7 (C-3).

α-Homoallonojirimycin (α-allo-HNJ) (9) HRFABMS m/z 194.1024 [M+H]⁺(C₇H₁₆NO₅ requires 194.1028). ¹³C-NMR (100 MHz, D₂O) δ57.2 (C-5), 58.1(C-1), 62.7 (C-1′), 63.5 (C-6), 72.0 (C-4), 72.1 (C-3), 72.2 (C-2).

α-Homogalactonojirimycin (α-galacto-HNJ) (10) HRFABMS m/z 194.1028[M+H]⁺ (C₇H₁₆NO₅ requires 194.1028). ¹³C-NMR(100 MHz, D₂O), δ55.8 (C-5),59.3 (C-1), 59.6 (C-1′), 64.5 (C-6), 71.8 (C-2), 71.9 (C-4), 73.8 (C-3).

N-Methyl-1-deoxygalactonojirimycin (N-Me-DGJ) (11) HRFABMS m/z 178.1081[M+H]⁺ (C₇H₁₆NO₄ requires 178.1079). ¹³C-NMR (100 MHz, D₂O) δ44.2(N—CH₃), 62.9 (C-1), 63.6 (C-6), 68.5 (C-2), 69.7 (C-5), 73.0 (C-4),77.8 (C-3).

N-Ethyl-1-deoxygalactonojirimycin (N-Et-DGJ) (12) HRFABMS m/z 192.1237[M+H]⁺ (C8H₁₈NO₄ requires 192.1236). ¹³C-NMR (100 MHz, D₂O) δ10.7, 48.9(N-ethyl), 57.8 (C-1), 63.2 (C-6), 65.0 (C-5), 69.9 (C-2), 73.0 (C4),77.9 (C-3).

N-Propyl-1-deoxygalactonojirimycin (N-Pr-DGJ) (13) HRFABMS m/z 206.1392[M+H]⁺ (C9H₂₀NO₄ requires 206.1392). ¹³C-NMR (100 MHz, D₂O) δ13.9, 19.2,57.2 (N-propyl), 58.6 (C-1), 63.3 (C-6), 65.5 (C-5), 69.9 (C-2), 73.0(C4), 77.9 (C-3).

N-Butyl-1-deoxygalactonojirimycin (N-Bu-DGJ) (14) HRFABMS m/z 220.1546[M+H]⁺ (C₁₀H22NO₄ requires 220.1549). ¹³C-NMR (100 MHz, D₂O) δ16.1,23.0, 27.9, 55.0 (N-butyl), 58.6 (C-1), 63.3 (C-6), 65.5 (C-5), 69.9(C-2), 73.0 (C-4), 77.9 (C-3).

N-Hydroxyethyl-l-deoxygalactonojirimycin (N-HE-DGJ) (15) HRFABMS m/z208.1183 [M +H]⁺ (C₈H₁₈NO₅ requires 208.1185). ¹³C-NMR (100 MHz, D₂O)δ56.0 (N-CH₂—), 59.2 (C-1), 60.9 (N—CH₂CH₂OH), 63.7 (C-6), 66.4 (C-5),69.7 (C-2), 73.3 (C-4), 77.8 (C-3).

β-1-C-Butyl-deoxygalactonojirimycin (16) HRFABMS m/z 220.1543 [M+]⁺(C₁₀H₂₂NO₄ requires 220.1549). ¹³C-NMR(100 MHz, D₂O) (δ16.1, 25.0, 29.6,33.5 (C-butyl), 61.1 (C-5), 61.8 (C-1), 64.2 (C-6), 71.8 (C-4), 74.9(C-2), 77.9 (C-3).

Enzyme and in vitro enzyme assay α-Gal A was expressed from Sf-9 insectcells infected with a recombinant baculovirus encoding normal α-Gal Agene and purified to homogeneity by concanavalin A-Sepharose and Mono Q(Pharmacia LKB Biotechnology, Uppsala, Sweden) column chromatographyaccording to the published methods (37). The enzyme activity was assayedwith 2 mM p-nitrophenyl-α-D-galactoside as substrate in the presence ofbovine serum albumin (3 mg/ml) at pH 4.5.

Cell culture The Epstein-Barr virus-transformed lymphoblast lines from anormal adult and a Fabry patient with R301Q mutation in α-Gal A (38)were cultured in RPMI-1640 medium (Nissui Pharmaceutical Co., Tokyo,Japan) supplemented with 10% fetal calf serum (FCS) at 37° C. under 5%CO₂. Human fibroblasts from Gaucher and G_(M1)-gangliosidosis patientswere cultured in McCoy 5A medium supplemented with 10% FCS at 37° C.under 5% CO₂.

Intracellular α-Gal A assay Cells were cultured in the presence orabsence of inhibitor for 4 days. After being washed twice withphosphate-buffered -saline (PBS), the cells were harvested andhomogenized in 200 μl of H₂O, and 10 μl of the supernatant obtained bycentrifugation at 10,000 g was incubated at 37° C. with 50 μl of thesubstrate solution composed by 6 mM 4-methylumbelliferyl α-D-galactoside(4-MU-α-Gal) and 90 mM N-acetylgalactosamine in 0.1 M citrate buffer (pH4.5) for enzyme assay. One unit of intracellular enzyme activity wasdefined as one nmol of 4-methylumbelliferone released per hour at 37° C.

Intracellular galactosidase assay Cells were cultured in the presence orabsence of inhibitor for 5 days. After being washed twice with PBS, thecells were harvested and homogenized in 200 μl of H₂O, and 10 μl of thesupernatant obtained by centrifugation at 10,000 g was incubated at 37°C. with 50 μl of the substrate solution of 1 mM 4-methylumbelliferylβ-D-galactoside (4-MU-β-Gal) in 0.1 M citrate buffer (pH 4.5) for enzymeassay. One unit of intracellular enzyme activity was defined as one nmolof 4-methylumbelliferone released per hour at 37° C.

Intracellular glucocerebrosidase assay Cells were cultured in thepresence or absence of inhibitor for 5 days. After being washed twicewith PBS, the cells were harvested and homogenized in 200 μl of buffer Icomposed by 0.25% sodium taurocholate, 0. 1% Triton X-100 and 0.1 Mcitrate buffer (pH 5.2). The supernatant (10 μl) obtained bycentrifugation at 10,000 g was incubated at 37° C. with 50 μl of thesubstrate solution of 3 mM 4-methylumbelliferyl β-D-glucoside(4-MU-β-Glu) in the buffer I for determination of total β-glucosidaseactivity. The neutral β-glucosidase activity was determined byperforming the same assay except pre-incubation of the enzyme solutionwith 3 mM conduritol B epoxide (an irreversible inhibitor of acid β-Glu)at room temperature for 30 min. The glucocerebrosidase activity wasdetermined by subtracting the neutral β-glucosidase activity from thetotal enzyme activity. One unit of intracellular enzyme activity wasdefined as one nmol of 4-methylumbelliferone released per hour at 37° C.

EXAMPLE 1 In vitro Inhibition and Intracellular Enhancement of α-Gal Ain Fabry Lymphoblasts

Structural Basis of In Vitro Inhibition of α-Gal A

The summary of IC₅₀ and selected K_(i) values of DGJ and its derivativesare shown in Table 2.

TABLE 2 In vitro inhibition of α-Gal A by DGJ derivatives. InhibitorIC₅₀ and (K_(i))^(a) 1-deoxynojirimycin (DNJ) (1) 830 manno-DNJ (2)N.I.^(b) allo-DNJ (3) N.I. galacto-DNJ (DGJ) (4) 0.04 (K_(i), 0.04)gulo-DNJ (5) N.I. 2-deoxy-DGJ (6) 250 α-homonojirimycin (α-HNJ) (7) N.I.α-manno-HNJ (8) 464 α-allo-HNJ (9) 4.3 (K_(i), 2.6) α-galacto-HNJ(α-HGJ) (10) 0.21 (K_(i), 0.17) N-methyl-DGJ (11)  96 N-ethyl-DGJ (12)306 N-propyl-DGJ (13) 301 N-butyl-DGJ (14) 300 N-hydroxyethyl-DGJ (15)520 β-1-C-butyl-DGJ (16) 24 (K_(i), 16) IC₅₀ values (i.e. inhibitorconcentration giving 50% inhibition) were determined by variation ofinhibitor concentrations. K_(i) values were evaluated from the slope ofLineweaver-Burk plots. Assays were performed as described under“Methods.” All constants are expressed in micromolar. ^(a)Km of α-Gal Awas determined as 0.17 mM with p-nitrophenyl-α-D-galactopyranoside.^(b)Inhibition was less than 50% at 1000 μM.DGJ and its Isomers

DGJ (galacto-DNJ) was synthesized from D-glucose and found to be anextremely powerful inhibitor of coffee bean α-galactosidase (39). In thedevelopment of the present invention, both IC₅₀ and K_(i) values of DGJtoward human lysosomal α-Gal A were calculated to be 0.04 μM (Table 2,FIG. 3A). DNJ (1) was a weak inhibitor of this enzyme with an IC₅₀ valueof 830 μM, while other isomers such as manno-(2), allo-(3), and gulo-DNJ(5) showed no appreciable inhibition even at 1000 μM. The deoxygenationat C-2 of DGJ (6) reduced its inhibitory potential over 6000-fold. Theseresults suggested to Applicants that a galactosyl configuration of animino sugar is preferable for the inhibition of α-Gal A.

α-HNJ and Isomers

α-HNJ (7) was not an inhibitor of α-Gal A, but α-manno-HNJ (8) was aweak inhibitor of the enzyme. α-galacto-HNJ (10) mimickingα-D-galactopyranose was first expected to be a more specific and potentinhibitor of α-Gal A than DGJ. From ¹H-NMR studies, the³J_(H,H)-coupling constants (J_(2,3)=9.8 Hz, J_(3,4)=3.0 Hz, J_(4,5)=2.6Hz,) observed for α-galacto-HNJ (10) clearly showed that this compoundis predominantly in a chair conformation which maintained theground-state structure of the substrate. However, insertion of ahydroxymethyl group to the α-anomeric position of DGJ decreased theaffinity for α-Gal A by approximately 4-fold. Surprisingly, α-allo-HNJ(9) showed a fairly potent inhibitory activity toward α-Gal A, with anIC₅₀ value of 4.3 μM. From its structure, this compound could form twodifferent conformations as shown in FIG. 2, α-allo-HNJ (FIG. 2A) vs. C-2epimer of α-galacto-HNJ (FIG. 2B). The J_(H,H)-coupling constants incompound 9 (J_(1,2)=4.6 Hz; J_(2,3)=2.9 Hz; J_(3,4)=2.9 Hz; J_(4,5)=6.5Hz) indicated that the conformation deviates from a chair form as aresult of the 1,3 syn-diaxial interaction between the substituents atC-2 a n d C-4 (FIG. 2B). Furthermore, the C-5 carbon in the ¹³C-NMRspectrum of 9 is observed as a broad signal, presumably due to “wobble”at C-5. The potent inhibitory activity of α-allo-HNJ (9) toward α-Gal Amay be due to the partial stereochemical and conformational similaritiesbetween a flexible α-allo-HNJ conformation (FIG. 2C) and a galactosylcation (FIG. 2D), which has been presumed to be a transition stateintermediate in the enzyme-catalyzed galactoside hydrolysis (40).

N-Alkyl Derivatives of DGJ

The N-alkyl derivatives of DGJ were studied for α-Gal A inhibitionbecause N-alkylation of DNJ and α-HNJ resulted in analogues withincreased potency and substrate specificity on digestive α-glucosidasesand processing α-glucosidase I (41-44), and N-alkylation of DNJ and DGJincreased inhibitory potential toward glucosyltransferase (45, 46).However, N-alkylation of DGJ markedly lowered its inhibitory activitytoward α-Gal A (Table 3), suggesting that modification of the iminogroup is not preferred for inhibition of α-Gal A. The naturallyoccurring DGJ derivative, β-1-C-butyl-DGJ, has recently been isolatedfrom Adenophorae Radix as a potent inhibitor of coffee beanα-galactosidase with an IC₅₀ value of 0.71 μM (35). The IC₅₀ value forα-Gal A was determined to be 24 μM.

Inhibition Mode of DGJ and its Derivatives

The inhibition mode of four potent inhibitors of α-Gal A, DGJ (4),α-galacto-HNJ (10), α-allo-HNJ (9) and β-1-C-butyl-DGJ (16) werestudied. Lineweaver-Burk plots indicated that they are competitiveinhibitors of α-Gal A (FIG. 3). The calculated Ki values of DGJ,α-galacto-HNJ, α-allo-HNJ and β-1-C-butyl-DGJ were found to be 0.04 μM,0.25 μM, 2.6 μM, and 16 μM, respectively.

Intracellular Enhancement of α-Gal A by the Enzyme Inhibitors

As shown in FIG. 4B, those DGJ derivatives that showed high inhibitoryactivity toward α-Gal A were tested for enhancement of intracellularα-Gal A activity in R301Q lymphoblasts. Treatment with DGJ at 100 μM for4 days increased enzyme activity in R301Q lymphoblasts by about 14-foldreaching 49% of normal. Enzyme activity was increased 5.2-fold,2.4-fold, and 2.3-fold by cultivation with α-galacto-HNJ, α-allo-HNJ,and β-1-C-butyl-DGJ at 100 μM, respectively, while weak inhibitors suchas N-alkyl derivatives of DGJ showed only a slight enhancement effect at100 μM. The effectiveness of intracellular enhancement paralleled to thein vitro inhibitory activity (FIG. 4A), indicating that a potentinhibitor serves as an effective enhancer.

The enzyme activity in R301Q lymphoblasts was elevated with increasinginhibitor concentration in a certain range (for DGJ, 1-100 μM, FIG. 5).α-galacto-HNJ, α-allo-HNJ, and β-1-C-butyl-DGJ enhanced the α-Gal Aactivity by 12.5-, 3.9-, and 6.3-fold at 1000 μM, respectively. However,higher concentrations significantly reduced the enhancement effect,presumably causing inhibition of the enzyme activity. Applicantsconfirmed that inclusion of DGJ in the medium at 20 μM did not causeintracellular inhibition of globotriaosylceramide metabolism, indicatingthat intracellular DGJ concentration appeared to be lower than theconcentration normally required to inhibit the intracellular enzymeactivity at that condition (24). Intralysosomal enzyme activity may notbe inhibited by α-galacto-HNJ, α-allo-HNJ, and β-1-C-butyl-DGJ added inthe culture medium at 1000 μM, because these compounds exhibited weakerinhibitory activity than DGJ.

Although β-1-C-butyl-DGJ was a less effective inhibitor of α-Gal A thanα-allo-HNJ (K_(i)=16 μM vs K_(i)=2.6 μM), both enhancement effects werethe same at 100 μM, and the effect of β-1-C-butyl-DGJ at 1000 μM washigher than that by α-allo-HNJ at the same concentration (FIG. 5). Thissuggested that the bioavailability of β-1-C-butyl-DGJ may be better thanα-allo-HNJ, because increase of lipophilicity resulting from theC-alkylation at C-1 of DGJ may enhance the efficient transport acrosscell and ER membranes.

Calystegine compounds are polyhydroxylated nortropane alkaloids. Certainof these alkaloids exhibit potent inhibitory activities againstglycosidases (47). The enzyme activity in R301Q lymphoblasts was alsoelevated with increasing concentration of calystegine A₃ (FIG. 6A),calystegine B₂ (FIG. 6B), N-methyl-calystegine A₃ (FIG. 6C), andN-methyl-calystegine B₂ (FIG. 6D), respectively in a range of 100-1000μM.

The above results further supported Applicants' therapeutic concept thatpotent competitive inhibitors can serve as efficient chemical chaperonesto enhance intracellular mutant enzyme activity in cells derived frompatients of Fabry disease. According to this theoretical concept, morepotent inhibitors serve as more powerful chemical chaperones. As shownin the following examples, this therapeutic strategy of using potentcompetitive inhibitors or substrate analogs is not limited to Fabrydisease, but also applicable to other lysosomal storage disorders andgeneral hereditary disorders resulted from protein folding defects, suchas, but not limited to, α₁-antitrypsin deficiency, familialhypercholesterolemia, Alzheimer's disease, Marfan syndrome, osteogenesisimperfecta, carbohydrate-deficient glycoprotein syndrome, andMaroteaux-Lamy syndrome.

EXAMPLE 2 Intracellular Enhancement of β-galactosidase Activity inFibroblasts from G_(M1)-gangliosidosis Patients

G_(M1)-gangliosidosis is a progressive neurological disease caused byhereditary deficiency of lysosomal acid β-galactosidase (β-Gal) whichhydrolyses the terminal β-galactosidic residual of ganglioside G_(M1)and other glycoconjugates (27). Three clinical forms are described asinfantile type (severe form), juvenile type (sub-severe type), and adultonset type (mild type). No treatment is available for this disorder.

Applicants applied the strategy of using potent inhibitors as chemicalchaperones to enhance intracellular mutant enzyme activity to humanG_(M1)-gangliosidosis fibroblasts.

Human G_(M1)-gangliosidosis fibroblasts were cultured for 5 days withDGJ (4) and 4-epi-isofagomine (21) (both are inhibitors of β-Gal) at 500μM, and 50 μM, respectively (FIG. 6). The enhancement effect was notefficient with the fibroblasts from patients of infant type disease(BGF-1 and BGF-6). However, the intracellular enzyme activities infibroblasts established from patients diagnosed as juvenile and adulttypes disease were elevated to 9-53% of normal (FIG. 7B). The residualenzyme activity in BGF-7 was markedly increased 27-fold by inclusion ofcompound 4 at 500 μM (FIG. 7A). These results indicate that compound 4and 21 are powerful chemical chaperones for β-Gal, and can be used aspotential therapeutic agents for treatment of G_(M1)-gangliosidosis.

EXAMPLE 3 Intracellular Enhancement of Glucocerebrosidase Activity inFibroblasts from Gaucher Patients

Gaucher disease is characterized by the accumulation of glucosylceramide(glucocerebroside) due to the deficient activity of lysosomal acidβ-glucosidase (glucocerebrosidase, β-Glu) (26). Three types of Gaucherdisease have been identified: 1) type 1 (adult-onset), lack of primarycentral nervous system involvement; 2) type 2 (infantile-onset), acuteneuronopathic form of the disease with an early onset; type 3(late-infantile/juvenile-onset), subacute neuronopathic form. Enzymereplacement therapy is effective only for type 1 disease.

In Vitro Inhibition of Glucocerebrosidase

Various natural and synthetic compounds were tested with human normalβ-Glu for inhibitory activity, and the IC₅₀ values are shown in Table 3.

TABLE 3 In vitro inhibition of human β-glucocerebrosidase. InhibitorIC₅₀ Calystegine A₃ (17) 3.1 Calystegine A₅ (22) 31 Calystegine B₁ (23)2.5 Calystegine B₂ (18) 0.99 Calystegine B₃ (24) 76 Calystegine B₄ (25)82 Calystegine C₁ (26) 2.5 N-Metyl-calystegine B₂ (20) 320 DMDP (27) 280DAB (28) 160 Castanospermine (29) 19 DNJ (1) 240 N-Butyl-DNJ (30) 270N-Dodecyl-DNJ (31) 0.05 DNJ bisulfite (32) 28 Isofagomine (33) 0.04N-Butyl-isofagomine (34) 44 N-(3-cyclohexylpropyl)-isofagomine (35) 100N-(3-phenylpropyl)-isofagomine (36) 69 N-[(2E, 6Z, 10Z)-3,7,11- 1.5trimethyldodecatrienyl]-isofagomine (37) IC₅₀ values were determined byvariation of inhibitor concentrations. Assays were performed withglucocerebrosidase in 0.1 M citrate buffer (pH 5.2) using 4-MU-β-Glu assubstrate. All constants are expressed in micromolar.

Several potent inhibitors were found among calystegine compounds.Calystegine B₂ (18) (IC₅₀ value, 0.99 μM), calystegine B₁ (23) (2.5 μM),calystegine C₁ (26) (2.5 μM), and calystegine A₃ (17) (3.1 μM) were thebest inhibitors in this class. Castanospermine (29) is a known potentinhibitor for α-glucosidase, however, it also present fair inhibitoryactivity against β-Glu (19 μM). DNJ (1) and N-butyl-DNJ (30) were weakinhibitors for this enzyme, however, N-dodecyl-DNJ (31) turned to be oneof the most potent inhibitor with IC₅₀ at 0.05 μM. Since DNJ andN-butyl-DNJ were moderate inhibitors of the enzyme, the high potency ofthis compound (31) is believed from the long alkyl chain in themolecular which is probably recognized by the recognition domainnormally recognizing the ceramide part of the substrate. Isofagomine(IFG, 33) was reported as a potent inhibitor against almondβ-galactosidase (40), and revealed as the most potent inhibitor amongthose tested with IC₅₀ value at 0.04 μM. Modification of the imino group(compounds 34-37) of IFG reduced inhibitory activity substantially. Thisresult consistent with Applicants' earlier finding with α-Gal A in whichalkyl modification of DGJ nullified its inhibitory activity. Noticeably,compound 37 which contains a 12 carbon chain in the backbone increased30-fold in its potency compared with compound 32 which contains a 4carbon chain. Combined with the result generated from DNJ (1) andN-dodecyl-DNJ (31), it is expected that N-dodecyl-IFG serves as apowerful inhibitor for human β-Glu. In accordance with the invention,these inhibitors should be effective in enhancing activity of thedefective enzyme associated with Gaucher disease and treatment of thedisorder.

Intracellular Enhancement of β-Glu Activity in Fibroblasts from GaucherPatients

Isofagomine and derivatives IFG (33) is the most potent inhibitor testedfor β-Glu in vitro. Its intracellular enhancement activity wasinvestigated with fibroblasts established from a Gaucher patient withN370S/N370S genotype. The intracellular enzyme activity was increased55-80% by cultivation the cell with IFG added in the culture medium at1-50 μM (FIG. 8A). Higher than 50 μM concentration nullified theenhancement effect. The enhancement effect was monitored for 5 days. Theresidual enzyme activity in Gaucher cells did not change on day 1 or 2,however, the enzyme activity was elevated after day 3 and increased morethan 80% at day 5 (FIG. 8B). This data demonstrated that IFG, a potentinhibitor of β-Glu, also serves as an enhancer for residual β-Glu in thecells derived from Gaucher patients when a appropriate concentration isapplied. Effective concentrations are expected to be lower than thoseneeded to inhibit the enzyme, but will be able to be determined throughroutine experimentation by those of skill in the art for Gaucher diseaseand other disorders. IFG derivatives (compounds 34-37) demonstratedsignificant impact on enhancement of the intracellular enzyme activityin Gaucher cells (N370S/N370S) cultivated with these compounds (FIG. 9).The residual enzyme activity was elevated 73% (compound concentration at10 μM) and 56% (100 μM) by compound 34, 106% (10 μM) and 47% (100 μM) bycompound 35, and 50% (10 μM) and 54% (100 μM) by compound 36,respectively. The residual enzyme activity was increased 43% bycultivation with compound 37 at 10 μM, however, decreased 53% with thecompound at 100 μM. Although the inhibitory activity of the IFGderivatives was weaker than IFG, the intracellular enhancement activityof the IFG derivatives appears to be higher than IFG, since theyachieved higher elevation of the mutant enzyme activity at lowerconcentrations. It is believed that the bioavailability of thesecompounds is significantly improved by the hydrophobic nature of themolecule, leading to easier crossing of cell and the ER membranes,thereby increasing the intracellular concentration of these compounds.Particularly, compound 37 at 100 μM decreased the residual activity,presumably intracellular concentration reached to the concentrationrequired for inhibition.

N-dodecyl-DNJ N-dodecyl-DNJ (31) is one of the most potent inhibitors ofβ-Glu tested, and is believed to be recognized by the domain usuallyrecognizing ceramide of the natural substrate. N-dodecyl-DNJ alsoenhanced β-Glu activity in fibroblasts derived from Gaucher patient withN370S/N370S mutation. The enzyme activity increased 95% by cultivationthe cells with N-dodecyl-DNJ at 0.5 μM for 5 days (FIG. 10A). Theelevation of enzyme activity was dose-dependent between theconcentrations of 0.05-0.5 μM added to the medium. However,N-dodecyl-DNJ at higher than 1 μM nullified the enhancement effect. Thetime course of cultivation of the cells with N-dodecyl-DNJ at 0.5 μMindicated that the residual enzyme activity increased after day 3 (FIG.10B). Since N-dodecyl-DNJ and IFG are recognized by differentrecognition domains of the enzyme (N-dodecyl-DNJ, ceramide recognitiondomain vs. IFG, glucoside recognition domain), a compound with acombination of N-dodecyl-DNJ and IFG such as N-dodecyl-IFG is expectedto be a powerful agent for enhancing residual enzyme activity in Gauchercells.

Calystegine compounds Calystegine A₃ (17), calystegine B₁ (23),calystegine B₂ (18) and calystegine C₁ (26) exhibited potent inhibitoryactivity against β-Glu and were tested for intracellular enhancement ofβ-Glu activity with fibroblasts derived from Gaucher patient with agenotype of L444P/L444P (FIG. 11). The residual enzyme activity in thepatient's cells was increased 230%, 76%, 126% and 136% by cultivationwith calystegine B₂, B₁, A₃ and C₁ at 10 μM, respectively. The resultsindicate that these compounds also act as effective enhancers forGaucher fibroblasts.

Applicants have shown that i) α-allo-HNJ (9), α-HGJ (10),β-1-C-butyl-DGJ (16), calystegine A₃ (17), calystegine B₂ (18), N-metylcalystegine A₃ (19), and N-methyl calystegine B₂ (20) are able toeffectively increase the intracellular α-Gal A activity in Fabrylymphoblasts by cultivation the cells with the above individual compoundin concentration ranges of 10-1000 μM; ii) DGJ (4) and 4-epi-isofagomine(21) are able to effectively enhance the intracellular β-Gal activity inG_(M1)-gangliosidosis fibroblasts by cultivation the cells with theabove individual compound in concentration ranges of 50-500 μM; iii)Calystegine B₂ (18), calystegine B₁(23), calystegine A₃ (17),calystegine C₁ (26), N-dodecyl-DNJ (31), isofagomine (33),N-butyl-isofagomine (34), N-(3-cyclohexylpropyl)-isofagomine (35),N-(3-phenylpropyl)-isofagomine (36) andN-[(2E,6Z,10Z)-3,7,11-trimethyldodecatrienyl]-isofagomine (37) are ableto effectively enhance the intracellular β-Glu activity in Gaucherfibroblasts by cultivation the cells with the above individual compoundin concentration ranges of 0.05-100 μM.

Applicants earlier disclosed in U.S. application Ser. No. 09/087,804 amethod for treatment of Fabry disease by administration of potentcompetitive inhibitors of α-Gal A to enhance the intracellular α-Gal Aactivity in the Fabry lymphoblsts. The mechanism underlying thistreatment is believed to be that the competitive inhibitors serve aschemical chaperones to induce/ensure the proper (native) conformation ofthe mutant protein for a smooth escape from the ER quality controlsystem, thus accelerate the maturation and transport leading to increaseof the intracellular enzyme activity. In the present application,Applicants further demonstrate the correlation between in vitroinhibition and intracellular enhancement with a series of inhibitors forα-Gal A. The results clearly show that more potent competitiveinhibitors serve as more powerful enhancers for the mutant enzyme.

In the present application, it is demonstrated that the method fortreatment of Fabry disease by administration of potent competitiveinhibitor of the defective enzyme can be applied toG_(M1)-gangliosidosis and Gaucher disease, both diseases belong tolysosomal storage disorder family, and potent inhibitors for β-Gal orβ-Glu can effectively enhance the intracellular enzyme activities in thefibroblasts established from patients of G_(M1)-gangliosidosis orGaucher disease, respectively. Since the lysosomal storage disordersshare the same biological and biochemical pathogenic mechanisms, thismethod of using potent competitive inhibitors of the defective enzyme isexpected to be applicable for the treatment of other lysosomal storagedisorders listed in Table 1.

Recent study on glycosidase inhibitors showed that conventional type ofiminosugars having a nitrogen atom replacing the ring oxygen of a sugarsuch as 1-deoxynojirimycin are more potent and selective inhibitors forα-glycosidases, whereas 1-N-iminosugars having a nitrogen atom at theanomeric position of the pyranose ring are more potent and selective forβ-glycosidases (40). We expect that conventional type of iminosugars(nojirimycin type), e.g., 1-deoxy-nojirimycin,1-deoxy-galactonojirimycin, 1-deoxy-iduronojirimycin, 1,2-didcoxy-2-N-acetamido-nojirimycin, 1-deoxymannonojirimycin, 1-deoxyfuconojirimycin,2,6-dideoxy-2,6-imino-sialic acid, and1,2-dideoxy-2-N-acetamido-galactonojirimycin (FIG. 12), which have aground-state structure of the substrate of a defective enzyme, e.g.,α-glucosidase, α-galactosidase, α-L-iduronidase,α-N-acetylglucosaminidase, α-mannosidase, α-L-fucosidase,α-N-acetyl-neuraminidase, and α-N-acetylgalactosaminidase, are potentinhibitors and powerful enhancers for treatment of Pompe disease, Fabrydisease, Hurler-Scheie disease, Sanfilippo disease, α-mannosidosis,Fucosidosis, Sialidosis and Schindler-Kanzaki disease, respectively. Wealso expect that 1-N-iminosugars, e.g., isofagomine, 4-epi-isofagomine,2-N-acetamido-isofagomine, 6-carboxy-isofagomine, and2-hydroxy-isofagomine (FIG. 12), which have a ground-state structure ofthe substrate of a defective enzyme, e.g., β-glucosidase,β-galactosidase, β-N-acetylglucosaminidase, β-glucuronidase andβ-mannosidase, are potent inhibitors and powerful enhancers fortreatment of Gaucher disease, G_(M1)-gangliosidosis, Krabbe disease,Morquio disease, Tay-Sachs disease, Sandohoff disease, Sly disease, andβ-mannosidosis. A summary of potent competitive inhibitors which areexpected to effectively enhance the mutant enzyme activity associatedwith lysosomal storage disorders is presented in Table 4.

TABLE 4 Summary of expected enhancers for lysosomal storage disorders.Disorder Targeting enzyme Enhancer Ref. Pompe disease α-glucosidase1-deoxynojirimycin (DNJ) 28 α-homonojirimycin 31 castanospermine *11Gaucher disease Acid β-glucosidase, or isofagomine 40 glucocerebrosidaseN-dodecyl-DNJ *1 calystegines A₃, B₁, B₂, C₁ *1 Fabry diseaseα-galactosidase A 1-deoxygalactonojirimycin 24 α-allo-homonojirimycin *2α-galacto-homonojirimycin *2 β-1-C-butyl-deoxynojirimycin *2calystegines A₃, B₂ *1 N-methyl calystegines A₃, B₂ *1G_(M1)-gangliosidosis Acid β-galactosidase 4-epi-isofagomine *11-deoxygalactonojirimycin *1 Krabbe disease Galactocerebrosidase4-epi-isofagomine 40 1-deoxygalactonojirimycin 28 Morquio disease B Acidβ-galactosidase 4-epi-isofagomine 40 1-deoxygalactonojirimycin 28α-Mannosidosis Acid α-mannosidase 1-deoxymannojirimycin 40 Swainsonine*3 Mannostatin A *4 β-Mannosidosis Acid β-mannosidase2-hydroxy-isofagomine 40 Fucosidosis Acid α-L-fucosidase1-deoxyfuconojirimycin *5 β-homofuconojirimycin *52,5-imino-1,2,5-trideoxy-L-glucitol *5 2,5-dideoxy-2,5-imino-D-fucitol*5 2,5-imino-1,2,5-trideoxy-D-altritol *5 Sanfilippo disease Bα-N-Acetylglucosaminidase 1,2-dideoxy-2-N-acetamido-nojirimycin 40Schindler-Kanzaki α-N- 1,2-dideoxy-2-N-acetamido- 40 diseaseacetylgalactosaminidase galactonojirimycin Tay-Sachs diseaseβ-Hexosaminidase A 2-N-acetylamino-isofagomine 401,2-dideoxy-2-acetamido-nojirimycin *6 nagstain and its derivatives *7,*8 Sandhoff disease β-Hexosaminidase B 2-N-acetamido-isofagomine 401,2-dideoxy-2-acetamido-nojirimycin *6 nagstain and its derivatives *7,*8 Hurler-Scheie disease α-L-Iduronidase 1-deoxyiduronojirimycin 402-carboxy-3,4,5-trideoxypiperidine *9 Sly disease β-Glucuronidase6-carboxy-isofagomine 40 2-carboxy-3,4,5-trideoxypiperidine *9Sialidosis Sialidase 2,6-dideoxy-2,6-imino-sialic acid 40 Siastatin B*10 References: *1, This application. *2, Asano, N., Ishii, S., Kizu,H., Ikeda, K., Yasuda, K., Kato, A., Martin, O. R., and Fan, J. -Q.(2000) In vitro inhibition and intracellular enhancement of lysosomalα-galactosidase A activity in Fabry lymphoblasts by1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem., inpress. *3, Dorling, P. R., Huxtable, C. R., and Colegate, S. M. (1980)Inhibition of lysosomal α-mannosidase by swainsonine isolated fromSwainsona canescens. Biochem. J. 191, 649-651. *4, Aoyagi, T., Yamamoto,T., Kojiri, K., Morishima, H., Nagai, M., Hamada, M., Takeuchi, T., andUmezawa, H., (1989) Mannostatins A and B: new inhibitors ofalpha-D-mannosidase, produced by Streptoverticillium verticillus var.quintum ME3-AG3: taxonomy, production, isolation, physico-chemicalproperties and biological activities. J. Antibiot. 42, 883-889. *5,Asano, N., Yasuda, K., Kizu, H., Kato, A., Fan, J. -Q., Nash, R. J.,Fleet, G. W. J., and Molyneux, R. J. (2000) Novel α-L-fucosidaseinhibitors from the bark of Angylocalyx pynaertii (Leguminosae).Submitted. *6, Fleet, G. W. J., Smith, P. W., Nash, R. J., Fellows, L.E., Parekh, R. J., and Rademacher, T. W. (1986) Synthesis of2-acetamido-1,5-imino-1,2,5-trideoxy-D-mannitol and of2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol, a potent and specificinhibitor of a number of β-N-acetylglucosaminidases. Chem. Lett.1051-1054. *7, Aoyagi T, Suda H, Uotani K, Kojima F, Aoyama T, HoriguchiK, Hamada M, Takeuchi T (1992) Nagstatin, a new inhibitor ofN-acetyl-beta-D-glucosaminidase, produced by Streptomyces amakusaensisMG846-fF3. Taxonomy, production, isolation, physico-chemical propertiesand biological activities. J Antibiot (Tokyo) 45, 1404-8. *8, Tatsuta K,Miura S, Ohta S, Gunji H (1995) Syntheses and glycosidase inhibitingactivities of nagstatin analogs. J Antibiot (Tokyo) 48, 286-8. *9, Cencidi Bello I, Dorling P, Fellows L, Winchester B (1984) Specificinhibition of human β-D-glucuronidase and α-L-iduronidase by atrihydroxy pipecolic acid of plant origin. FEBS Lett 176, 61-4. *10,Umezawa H, Aoyagi T, Komiyama T, Morishima H, Hamada M (1974)Purification and characterization of a sialidase inhibitor, siastatin,produced by Streptomyces. J Antibiot (Tokyo) 27, 963-9. *11, Pili R,Chang J, Partis RA, Mueller RA, Chrest FJ, Passaniti A (1995) Thealpha-glucosidase I inhibitor castanospermine alters endothelial cellglycosylation, prevents angiogenesis, and inhibits tumor growth. CancerRes 55, 2920-6.

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1. A method for increasing in a mammalian cell the activity ofacid-β-galactosidase, which method comprises contacting the cell with areversible competitive inhibitor of the acid-β-galactosidase in anamount effective to increase acid-β-galactosidase activity.
 2. Themethod of claim 1, wherein the competitive inhibitor is selected fromthe group consisting of 4-epi-isofagomine and 1-deoxygalactonojirimycin.3. The method of claim 2, wherein the competitive inhibitor is1-deoxygalactonojirimycin.
 4. The method of claim 2, wherein thecompetitive inhibitor is 4-epi-isofagomine.